1. Field of the Invention
The present invention relates to a novel reverse transcriptase composition having improved storage stability. More particularly, the present invention is concerned with a reverse transcriptase composition having improved storage stability, comprising a reverse transcriptase, an effective stabilizing amount of at least one organic stabilizing reagent selected from the group consisting of trehalose and a nucleic acid containing a transcriptional initiation site recognizable by the reverse transcriptase, and an effective stabilizing amount of a metal salt capable of producing bivalent positive ions in an aqueous solution of the metal salt. The present invention is also concerned with a method for improving storage stability of a reverse transcriptase, which comprises adding to a reverse transcriptase an effective stabilizing amount of at least one organic stabilizing reagent selected from the group consisting of trehalose and a nucleic acid containing a transcriptional initiation site recognizable by the reverse transcriptase, and an effective stabilizing amount of a metal salt capable of producing bivalent positive ions in an aqueous solution of the metal salt.
Conventionally, for maintaining the stability of a reverse transcriptase, it has been necessary to store the reverse transcriptase at a temperature as low as -20 to -80.degree. C. According to the present invention, however, a reverse transcriptase can be stably stored in the form of the above-mentioned composition for a prolonged period of time at a temperature up to at least 4.degree. C. without suffering denaturation of the reverse transcriptase. Further, by virtue of such a relatively high temperature usable for stable storage, the viscosity of the reverse transcriptase composition of the present invention can be advantageously maintained at a low level, so that it becomes possible to accurately dispense the composition by a quantity corresponding to a desired enzyme activity, thereby achieving high reproducibility in experiments using the reverse transcriptase.
The reverse transcriptase composition of the present invention, which has improved storage stability, can be advantageously used in the fields of genetic engineering and virology, with respect to various techniques in which the reverse transcriptases are employed. Particularly, in a method generally employed for the determination of a virus, in which a reverse transcriptase activity is used as an index, the transcriptase composition of the present invention can be advantageously used as a standard substance for determining the amount of virus.
2. Prior Art
Reverse transcriptase is an enzyme having the activity of synthesizing from an RNA template a DNA having a nucleotide sequence complementary to the RNA. This enzyme was discovered in the recent development of molecular biology. Examples of reverse transcriptases include those which are derived from avian myeloblast virus (AMV), Ras associated virus type 2 (RAV-2), mouse molony murine leukemia virus (M-MuLV) and human immunodefficiency virus type 1 (HIV-1). These reverse transcriptases are commercially readily available.
Further, on a laboratory scale, a reverse transcriptase can be easily obtained by directly separating the enzyme from a virus. For example, a reverse transcriptase can be easily prepared by treating particles of retroviruses, such as human immunodefficiency virus Type 2 (HIV-2), human T-cell leukemia virus type 1 (HTLV-1), simian AIDS virus (SIV), feline AIDS virus (FIV) and Rous related avian sarcoma virus, with a surfactant, such as Triton X-100 (manufactured and sold by Sigma Chemical Company, USA). The above-mentioned reverse transcriptases have been widely used in the synthesis of a cDNA from a messenger RNA.
Reverse transcriptase is also used in the PCR (polymerase chain reaction) method which is recently a most widely used nucleic acid amplification technique. Specifically, the PCR method is a technique to amplify at least one type of nucleic acid using a thermostable DNA polymerase and two types of primers specific to the nucleic acid, in which a cycle of DNA polymerase reaction and annealing is repeatedly performed by repeating the cycle of raising and lowering of the temperature of the reaction system. In the PCR method, the activity of the DNA polymerase is specific to the DNA. Therefore, when an RNA is used as a starting material for the nucleic acid amplification using the technique of the PCR method, it is necessary to employ a method in which a DNA is prepared from the RNA using a reverse transcriptase, and the obtained DNA is amplified by the PCR method such a method is called an "RT-PCR (reverse transcription polymerase chain reaction) method"!. For this reason, in the study of an RNA using the technique of the PCR method, the reverse transcriptase is indispensable. Particularly, in recent years, the number of studies made with respect to the determination of the amount of a virus, such as HIV, HCV or the like, in terms of the RNA amount has been increasing, so that the number of the test kits developed for use in such determination has also been increasing. Therefore, the reverse transcriptase is a commercially important enzyme.
Further, the reverse transcriptase is also employed in the LCR (ligase chain reaction) method which is another nucleic acid amplifying technique. Specifically, in the LCR method, the amplification of at least one type of nucleic acid is performed in substantially the same manner as in the PCR method, except that a thermostable DNA ligase is used instead of the thermo-stable DNA polymerase, in which the cycle of ligation reaction and annealing is repeatedly performed by repeating the cycle of raising and lowering of the temperature of the reaction system. In the LCR method also, the activity of the DNA ligase is specific to the DNA. Therefore, when an RNA is used as a starting material for the nucleic acid amplification using the technique of the LCR method, it is necessary to employ a method in which a DNA is prepared from the RNA using a reverse transcriptase, and the obtained DNA is amplified by the LCR method such a method is called an "RT-LCR (reverse transcription ligase chain reaction) method"!. For this reason, also in the study of an RNA using the technique of the LCR method, the reverse transcriptase is indispensable.
The reverse transcriptase is also used in NASBA (nucleic acid sequence based amplification) method. Specifically, the NASBA method is a method of amplifying a nucleic acid by using a reverse transcriptase, a DNA polymerase, a RNase H and two types of primers, in which the amplification of the nucleic acid is performed at room temperature by the cascade reaction of the enzymes. In this method also, the reverse transcriptase is indispensable for amplifying a nucleic acid.
On the other hand, in the field of virology, it has generally been practiced to determine the amount of virus from the activity of a reverse transcriptase contained in the virus, in which a reverse transcriptase is used as a standard substance. Examples of reverse transcriptases used in this field include those which are derived from retroviruses, such as human immunodefficiency virus type 1 (HIV-1), human immunodefficiency virus type 2 (HIV-2), human T-cell leukemia virus type 1 (HTLV-1), simian AIDS virus (SIV), feline AIDS virus (FIV) and Rous avian sarcoma virus.
These reverse transcriptases are generally stored at a temperature of from -20 to -80.degree. C. in the form of a solution thereof in a buffer comprising a monovalent or divalent metal salt (such as NaCl or MgCl.sub.2), an SH group-containing protective reagent (such as mercaptoethanol and/or dithiothreitol) and a high concentration (about 50% by volume) of glycerol.
Conventionally, for performing the reverse transcription reaction which is usually conducted at 37.degree. C., the preparatory operation prior to the reaction is generally conducted as follows. A container containing the reverse transcriptase is taken out from a freezer having a temperature as low as -20 to -80.degree. C. and placed under ambient temperature conditions of 4.degree. C. or more (the reverse transcriptase is unstable under such temperature conditions), followed by taking out a necessary amount of the reverse transcriptase from the container by dispensing. Then, the container is returned to the freezer having a temperature of from -20 to -80.degree. C. In this preparatory operation, the reverse transcriptase is necessarily left under the above-mentioned temperature conditions, at which the enzyme is unstable, for a prolonged period of time during the handling thereof. Therefore, as the number of such operations increases, the activity of the stored reverse transcriptase remaining in the container decreases. Therefore, it is difficult to maintain the activity of the reverse transcriptase at the same level for a prolonged period of time. For avoiding the lowering of the activity of the reverse transcriptase, which is caused by the repetition of the above operation, it has conventionally been practiced to conduct the operation of taking out the necessary amount of the solution containing the reverse transcriptase from the container within a short period of time inside of the freezer (having a temperature of from -20 to -80.degree. C.), followed by performing a reverse transcription reaction using the reverse transcriptase immediately after the above removing operation. However, in this technique, the temperature of the removed solution containing the reverse transcriptase is inevitably very low, so that the solution has high viscosity. Therefore, with this method, it is impossible to accurately dispense the solution in a quantity corresponding to a desired reverse transcriptase activity. Further, each of the above-mentioned conventional techniques is accompanied by the problem of a bad smell caused by the SH group-containing protective reagent used as a stabilizer (e.g., mercaptoethanol), which has an unfavorable influence on the person conducting the operation.
Further, with respect to the technique to stabilize the reverse transcriptase, a stabilizing method for the reverse transcriptase using a hybridization product of a template-primer represented by (rA).sub.n (dT).sub.12-18 is known (see Biochemical and Biophysical Research Communication Vol 167, (2), pp 673 (1990)!. However, in this method, the enzyme activity retaining ratio as measured after the enzyme is stored at 4.degree. C. for 300 hours is as low as 70%, so that satisfactory storage stability cannot be achieved by this method.
As can be seen from the above, in experimental studies using the reverse transcriptase, it has conventionally been difficult to conduct experiments with high reproducibility, due to the instability of the reverse transcriptase.
Due to a recent sudden increase in the number of patients infected with HIV, there has been a rapid increase in the number of testings for measuring the reverse transcriptase activity which is used for determining the amount of virus. In the testings, the reverse transcriptase is used as a standard substance. However, it has conventionally been difficult to measure the reverse transcriptase activity with respect to many samples with high reproducibility, due to the instability of the reverse transcriptase. Especially when molecular biological experiments using a reverse transcriptase must be conducted for a long period of time, various disadvantages are likely to occur. That is, during the repetition of the operation of taking out the reverse transcriptase from a container stored in a freezer or the like and returning the container to the freezer or the like, the reverse transcriptase remaining in the container is caused to be denaturated. Therefore, since such a remaining denaturated enzyme can no longer be used, it becomes necessary to replace the denaturated enzyme with a newly purchased or newly prepared reverse transcriptase to continue the experiments. This is disadvantageous from the viewpoint of energy, cost and time.
For improving the storage stability of the reverse transcriptase, it has been proposed to use the reverse transcriptase in a freeze-dried form which is more stable than in an aqueous solution form. However, in the conventional storage technique for the reverse transcriptase, a reverse transcriptase solution for storage contains glycerol in a high concentration. Therefore, due to the high concentration of glycerol in the solution for storage, it is difficult to obtain the reverse transcriptase in a freeze-dried form, so that the stability of the reverse transcriptase for a prolonged period of time has been unable to be realized.
If the stability of the reverse transcriptase can be successfully improved such that the storage stability of the reverse transcriptase at a temperature up to at least 4.degree. C. can be maintained for a prolonged period of time, it would become unnecessary to use a freezer specially designed for storing the reverse transcriptase at a temperature as low as -20 to -80.degree. C. It would lead to a possibility of reduction in the cost for the above-mentioned virological studies and experiments.
Further, if the above-mentioned improved storage stability is achieved, it would become possible to lower the viscosity of the solution containing the reverse transcriptase, so that it would become possible to accurately dispense the composition by a quantity corresponding to a desired enzyme activity, thereby achieving high reproducibility in experiments using the reverse transcriptase. The achievement of such an improved storage stability of the reverse transcriptase is advantageous especially when the reverse transcriptase is used as a standard substance in the determination of the amount of virus, in which the reverse transcriptase activity is used as an index. Further, by virtue of the high reproducibility of experiments using a reverse transcriptase, it becomes possible to analyze a large number of samples by the use of a reverse transcriptase. This is advantageous in view of the recent tendency that the numbers of the samples to be analyzed by the use of a reverse transcriptase is increasing.
Therefore, in various techniques using a reverse transcriptase, it has been strongly desired to improve the storage stability of a reverse transcriptase, so that experiments using a reverse transcriptase can be conducted with high reproducibility and at low cost.